The ability to measure directly forces between biopolymers in macroscopic condensed arrays has greatly changed our understanding of how molecules interact at close spacings, the last 1-1.5 nanometers separation. The universality of the force characteristics observed for a wide variety of macromolecules, including DNA, proteins, lipid bilayers, and carbohydrates, and for the exclusion of small solutes and salts from macromolecular surfaces has led us to conclude that the energy associated with changes in structuring water between surfaces dominates intermolecular forces. [unreadable] DNA-DNA attractions: DNA packaging by multivalent ions is a critical testing ground for understanding forces between charged molecules. If a sufficient concentration of multivalent ions is present, DNA will spontaneously assemble into an ordered array. The helices do not collapse to touching but are rather separated by 0.5-1.5 nm of solvent depending on the nature of the condensing ion. Attractive and repulsive forces balance at the equilibrium spacing. By combining the osmotic stress, pushing experiments with single molecule, magnetic tweezers, pulling experiments, we were able to separate the attractive and repulsive free energies at the equilibrium spacing for the commonly used condensing agents cobalt hexa-ammine, the biogenic alkylamines spermidine and spermine, and a synthetic +6 charged alkyl hexa-amine (essentially two spermidines joined by a butyl linker). The results confirmed our previous hypotheses for hydration forces. The 0.2 nm decay length exponential repulsive force is the hydration equivalent of the image charge repulsive force in electrostatics. The hydration atmosphere extending from a solvated surface stabilizes water structuring at the surface. Disruption of the atmosphere simply by replacing water with another surface will lower hydration energies regardless of the water structuring on the other surface. Repulsion should depend predominately on the water structuring of groups on the DNA surface and perhaps on the mode of binding (phosphate backbone or grooves), but not on the correlations of these groups with apposing helices. The attractive force is a 0.4 nm decay length exponential force that results from the direct interaction of surface hydration structures. Perturbations in water structure around one surface due to the close presence of another surface can either weaken or strengthen hydration energies depending on the mutual structuring water. We postulated that the attractive force had the same exponential 0.4 nm decay length as observed previously for repulsive hydration forces, but that the force was now attractive because of correlations in complementary water structuring on apposing helices. [unreadable] We are examining two other sets of homologous compounds, arginine and lysine peptides, in order to confirm and expand on the alkylamine results. The same limiting 0.2 nm decay length exponential repulsion is observed for mono-arginine to hexa-arginine and poly-arginine series regardless of charge as was seen for the alkylamine series. The force amplitudes, however, are different for the alkylamine and arginine series. Repulsion depends on the hydration properties of the bound counterion. Since the equilibrium spacing decreases with larger charge, but repulsion is constant, attraction increases with charge. Since attraction is presumably caused by complementary patches on apposing helices, increased attraction indicates increased correlation between hydrated amine charges on one helix with phosphate groups on another. We observe that attractive energies for the alkylamine and arginine series vary linearly with the inverse of the cation charge. This is consistent with a constant loss in entropy from correlating a single molecule regardless of charge, but a gain in interaction energy that increases with the number of charges. [unreadable] We have also begun measuring the packaging forces of salmon protamine assembled DNA. Protamines are small, arginine-rich peptides used to package DNA in sperm heads. Protamine-DNA forces resemble polyarginine-DNA interactions. The attraction with protamine, however, corresponds to penta- or hexa-arginine, not the 21 charges actually present. The sensitivity of equilibrium spacings of protamine-DNA complexes to the anion of the added salt indicates that salt is not operating through a screening or over-charging mechanism to weaken attraction, but rather through anion binding to protamine to lower the net protein charge. This is consistent with our previous work on the resolubilization of DNA assemblies at high concentrations of spermidine and spermine. By measuring the change in interhelical spacing as a function of salt concentration at constant osmotic pressure, we can quantitate anion binding with the same Maxwell equations used for solute exclusion.[unreadable] EcoRI sliding rates: Many specific sequence DNA binding proteins locate their target sequence by first binding to DNA nonspecifically, then linearly diffusing along DNA until either the protein dissociates from the DNA or finds the recognition sequence. How much DNA the protein can explore depends on the relative rates of both diffusion along the DNA and dissociation of the nonspecifically bound complex. We have completed our measurements of the sliding rate of EcoRI along DNA. We calculate sliding rates from the ratio of dissociation rates of EcoRI from DNA fragments containing 1 and 2 specific binding sites. We use several different 2 site DNA fragments with varied distances between sites. The measurements take advantage of the very different sensitivities of the relative specific-nonspecific binding constant and the nonspecific dissociation rate to salt concentration and osmotic pressure. We vary the nonspecific complex dissociation rate over a 2000-fold range through the salt concentration in order to better fit the diffusion rate of nonspecifically bound complex. The overall dissociation rate of the single specific site fragment can be held constant by adjusting the osmotic pressure. We measure dissociation rates using the gel mobility shift assay coupled with a stop reaction protocol as we have done previously. The data for all the 2 site DNA fragments can be well fit by a single sliding rate. The one-dimensional diffusion coefficient of EcoRI is 1000-fold slower than diffusion of free protein in water. We hypothesize that the extra drag is due to transient breaking of charge-charge contacts between DNA and protein. To test this, we plan to measuring sliding rates for the closely related nuclease BamHI that has fewer charge-charge pairs.